Cloning, sequencing and expression of a comamonas cyclopentanone 1,2-monooxygenase-encoding gene in escherichia coli

ABSTRACT

Cyclopentanone 1,2-monooxygenase (CPMO) from  Comamonas  (previously  Pseudomonas ) sp. strain NCIMB 9872 carries out the second step of a degradation pathway that allows the bacterium to use cyclopentanol as a sole carbon source for growth. In the present invention there is reported the localization of the CPMO-encoding gene (cpnB) on a 4.3-kb SphI fragment, the determination of its sequence. The 550-amino acid CPMO polypeptide (M t , 62,111) encoded by the gene was found to have 36.5% identity with the sequence of cyclohexanone 1,2-monooxygenase (CHMO) of  Acinetobacter  sp. strain NCIMB 9871. The 62-kDa CPMO was expressed in  E. coli  as an IPTG-inducible protein.

TECHNICAL FIELD

The present invention relates to an isolated DNA encoding acyclopentanone monooxygenase (CPMO), or an enzymatically active portionthereof, and expression vector and a transformed cell containing theisolates DNA.

BACKGROUND OF THE INVENTION

Comamonas (previously Pseudomonas) sp. NCIMB 9872 was one of the fewmicroorganisms that have been characterized to produce a Baeyer-Villigermonooxygenase (BVMO; Griffin, M., et al., Biochem. J. 129:595-603, 1972;Griffin, M., et al., Eur. J. Biochem. 63:199-209, 1976; and Willetts,A., Trends in Biotech. 15:55-62, 1997; for a recent review). BVMOs areflavoproteins that mimic the classical Baeyer-Villiger organic chemicalreaction which is a peracid-catalyzed oxidation of a ketone to an esteror lactone. The use of enzyme substitutes for the production of lactonesin high yield and optical purity is an attractive feature in currenttrends of research and development toward replacing chemical methodswith biological alternatives (Stinson, S. C., Chem. Eng. News, 83-104,1998). To date, the best characterized BVMO enzyme is that ofcyclohexanone monooxygenase (CHMO) produced by Acinetobacter sp. NCIMB9871 (Stewart, J. D., Curr. Org. Chem. 2:195-216, 1998; Willetts, A.,Trends in Biotech. 15:55-62, 1997). This is also the only BVMO whosegene has been cloned and sequenced (Chen, et al., J. Bacteriol.170:781-789, 1988). Recently, this valuable resource was used toengineer a “designer yeast” in a whole-cell approach to effect a varietyof asymmetric Baeyer-Villiger oxidations (Stewart, J. D., et al., J. Am.Chem. Soc. 120:3541-3548, 1998).

It would be highly desirable to be provided with a new CPMO having anincreased enzymatic activity for growing cells in a medium containingcyclopentanol or cyclopentanone as sole carbon source.

SUMMARY OF THE INVENTION

One aim of the present invention is to provide a new CPMO having anincreased enzymatic activity for growing cells in a medium containingcyclopentanol or cyclopentanone as sole carbon source.

In accordance with the present invention there is provided an isolatedDNA encoding a cyclopentanone monooxygenase (CPMO), or an enzymaticallyactive portion thereof, the isolated DNA being characterized by theability to hybridize specifically with the complement of the DNArepresented in SEQ ID NO:8 under stringent hybridization conditions.

Also in accordance with the present invention, there is provided anisolated DNA, wherein it codes for a cyclopentanone monooxygenase(CPMO), and contains:

-   -   (1) the nucleic acid sequence of SEQ ID NO:8;    -   (2) a sequence corresponding to said nucleic acid sequence in        the scope of the degeneration of the genetic code; or    -   (3) a sequence hybridizing under stringent conditions with the        sequence from (1) or (2), and still coding for cyclopentanone        monooxygenase (CPMO).

Still in accordance with the present invention, there is provided anisolated DNA encoding a cyclopentanone monooxygenase (CPMO), or anenzymatically active portion thereof, said isolated DNA having SEQ IDNO:8.

The present invention further provides an isolated DNA expression vectorencoding an enzymatically active cyclopentanone monooxygenase (CPMO)comprising a DNA characterized by a sequence as set forth in SEQ IDNO:8, or a portion thereof, said portion encoding said CPMO, inexpressible form.

In accordance with the present invention, there is also provided arecombinant vector comprising the isolated DNA as described above,wherein the isolated DNA encodes cyclopentanone monooxygenase.

In a preferred embodiment of the present invention, the isolated DNA hasa nucleic acid sequence of SEQ ID NO:8 or which, due to the degeneracyof the genetic code, is a functional equivalent thereof.

Also in accordance with the present invention, there is provided arecombinant vector containing one or more copies of a recombinant DNAdescribed above.

The recombinant vector may be a prokaryotic vector. The recombinantvector may also be a plasmid.

Therefore, in accordance with the present invention, there is alsoprovided a biologically functional plasmid or viral DNA vector, whichcontains a DNA as described above.

The present invention also provide a host cell comprising a recombinantvector as described above.

Accordingly, there is also provided a cell transformed with aheterologous DNA expression construct encoding an enzymatically activecyclopentanone monooxygenase (CPMO) comprising a DNA characterized by asequence as set forth in SEQ ID NO:8, or a portion thereof, said portionencoding said CPMO, in expressible form.

The cell may be a prokaryotic cell or it may be E. coli.

Still in accordance with the present invention, there is also provided apurified cyclopentanone monooxygenase (CPMO) having:

-   -   a) an amino acid sequence as set forth in SEQ ID NO:5;    -   b) an amino acid sequence encoded by a nucleic acid sequence as        set forth in SEQ ID NO:8; or    -   c) an amino acid sequence encoded by a nucleic acid sequence        hybridizing to a nucleic acid sequence complementary to the        nucleic acid sequence of step b) above under stringent        conditions, said amino acid sequence encoded in step c) having a        same activity as the amino acid sequence in a).

The present invention also provides a recombinant cyclopentanonemonooxygenase (CPMO) having an enzymatic activity superior to the onefrom a native Pseudomonas, and more preferably twice superior.

The recombinant cyclopentanone monooxygenase (CPMO) may be prepared fromComamonas sp. NCIMB 9872. The recombinant cyclopentanone monooxygenase(CPMO) has preferably a sequence as set forth in SEQ ID NO:5.

A method for growing cells in vitro in presence of cyclopentanol orcyclopentanone as sole source of carbon, said method comprising thesteps of:

-   -   a) transforming a cell with the expression construct described        above; and    -   b) growing the cell of step a) under suitable conditions in a        medium containing cyclopentanol or cyclopentanone as a sole        source of carbon.

To increase this gene potential, according to the invention it isreported herein the cloning of a cyclopentanone monooxygenase(CPMO)-encoding gene (cpnB) from Comamonas (Pseudomonas) sp. NCIMB 9872,the determination of its DNA and surrounding sequence and expression ofCPMO activity and protein in E. coli.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the first two steps of cyclopentanol degradation byPseudomonas sp. NCIMB 9872;

FIG. 2 illustrates the genetic organization in Comamonas sp. NCIMB 9872in the Sphl fragment containing cyclopentanone monooxygenase-encodinggene (cpnB) and additional open reading frames;

FIG. 3 illustrates an alignment of the amino acid sequence of the CPMOof Comamonas sp. NCIMB 9872 with that of CHMO from Acinetobacter sp.NCIMB 9871 and a steroid monooxygenase (STMO) from Rhodococcusrhodochrous;

FIG. 4 illustrates a SDS-PAGE of crude extracts from E. coli (pCMP201);and

FIG. 5 illustrates CPMO-encoding gene, designated cpnB.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Cloning of the Comamoitas sp. NCIMB 9872 CPMO-Encoding Gene

Pseudomonas sp. NCIMB 9872 (henceforth strain 9872) identified as aComamonas by 16S rDNA sequencing in this study, was purchased from theNational Collections of Industrial and Marine Bacteria Ltd (NCIMB,Aberdeen, Scotland) and grown at 30° C. in Luria-Bertani (LB) broth(Sambrook, J., et al., Molecular cloning: a laboratory manual, 2nd ed.Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), ormineral salt medium (MSM), pH 7.0, containing 2 ml of cyclopentanone.The MSM recipe contains per liter: 1.0 g of NH₄NO₃, 1.5 g of KH₂PO₄, 1.5g of Na₂HPO₄, 0.2 g MgSO₄·7H₂O, 0.01 g of CaCl₂·2H₂O, 0.005 ofFeSO·7H₂O, 0.002 g of MnSO₄·4H₂O and 0.1 g of yeast extract. Agar wasadded to 1.5% for plates. Genomic DNA of strain 9872 was prepared by theMarmur method (Marmur, J., J. Mol. Biol. 3: 208-218, 1961). At first, aSouthern hybridization of DNA digested with BamHI was carried out usingthe Acinetobacter NCIMB 9871 CHMO-containing gene as probe. Since therewas no positive result (hybridization conditions carried out at 65° C.)the CPMO protein was purified in order to obtain an N-terminal aminoacid sequence. The purification of CPMO protein fromcyclopentanone-grown cells was according to Griffin and Turgill(Griffin, M., et-al., Eur. J. Biochem. 63:199-209, 1976). Using anautomated protein sequencer (Perkin-Elmer model 477) a 40-residueamino-terminal sequence of the purified CPMO was obtained (FIG. 2). Thissequence, longer by 11 amino acids, is in perfect agreement with thatreported previously from the same organism (Willetts, A., Trends inBiotech. 15:55-62, 1997). Two degenerate oligodeoxynucleotide primers(5′-ACIACIATGA CIACNATGAC-3′ (SEQ ID NO:1) and 5′-ARRTGRTAIA RYTGRTA-3′(SEQ ID NO:2), corresponding to amino acids 2-8 and 35-40, respectively)were synthesized to amplify a 116-bp product from total DNA preparedfrom strain 9872. The PCR amplification was performed in a PerkinElmer-Model 2400 Thermal Cycler™ and the amplification conditions were94° C. for 1 min, 50° C. for 1 min and 72° C. for 1 min for 30 cycles.The amplified product was cloned directly in the pXcmkn12 vector (Cha,J., et al., Gene 136, 369-370, 1993), transformed in E. coli JM109 andthe resulting plasmid was designated pCMP10. Before using the amplifiedproduct as a gene probe its nucleotide sequence was confirmed.Nucleotide sequencing was determined by the Taq DyeDeoxy terminatorcycle sequencing kit and the ABI Prism 310 Genetic Analyzer (PerkinElmer). Plasmid isolation was performed by the method of Birnboim andDoly (Birnboim, H. C., and J. Doly, DNA. Nucleic Acids Res. 7:1513-1523,1979).

In FIG. 2, Orf1 is most likely a transcriptional activator of theNtrC-type, (Morett, E., L. Segovia, J. Bacteriol. 175:6067-6074, 1993).The amino acid sequence of ORF1 (C-terminal 391-amino acids) showed38-40% identity to equivalent regions of proteins such as NTRC_ECOLI(Nitrogen regulation protein NR(I) from E. coli; Miranda, et al., Thecomplete nucleotide sequence of the gInALG operon of Eschericha coli K1215:2757-2770,1987), ACOR_ALCEU (Acetoin catabolism regulatory proteinfrom Ralstonia eutropha; Kruger, N., et al., J. Bacteiol. 179:4391-4400,1992). The amino acid sequence of ORF2, showing similarity to enzymes ofthe short-chain alcohol dehydrogenase family (Jornvall, H., et al.,Biochemistry 34: 6003-6013, 1995), is most homologous (45-46% identity)to a putative oxidoreductase CY39.16C of Mycobacterium tuberculosis(Swiss Prot sp:Q10855) and fadG3 of M. tuberculosis (GenBank accessionnumber Z74025). For Pseudomonas sp. strain HI-201 the IacZ-Km^(r)cassette from pKOK6.1 (Kokotek, W., et al., Gene 84: 467-471, 1989) wasinserted into cpnB at the NsiI site.

In FIG. 2, the following terms are defined as follows: t_(fd),transcriptional termination sequence of phage fd; Km^(r), kanamycinresistance gene, IacZ, gene encoding b-galactosidase. Genes and markersare indicated with arrows.

To clone the CPMO-containing gene, the DNA insert from pCMP10 wasamplified, labeled by the digoxigenin-11-UTP system according tomanufacturer's instructions (Boehringer Mannheim GmbH) and used to probea Southern hybridization of strain 9872 genomic DNA digested withvarious restriction enzymes (BamHI, EcoRI, HindIII, KpnI, NheI, PstI,SaII, SphI and XbaI). As a result, a single hybridizing band of ca4.3-kb SphI fragment was obtained. Conditions of hybridization were asbefore. Subsequently, a purified 4.0- to 4.5-kb size fraction ofSphl-cut total DNA separated on a 0.8 % agarose gel was ligated to E.coli plasmid pUC18, which had been linearized and dephosphorylated. Aclone containing the 4.3-kb insert was screened by colony hybridizationusing the PCR product as a probe; this recombinant plasmid wasdesignated pCMP200.

DNA Sequence of the CPMO-Encoding Gene (cpnB) and the Flanking Region

Nucleotide sequencing of the CPMO-encoding gene was initiated by using aprimer designed from the sequence of the PCR product cloned in pCMP10and further extended using oligonucleotides derived from the newsequence. Both DNA strands of the SphI fragment were sequenced and foundto consist of 4281 base pairs (bp). The sequence was analyzed byGENETYX-Mac (Software Development Co., Ltd. Chiba, Japan) and the BLASTprogram (Altschul, S. F., et al., Nucleic Acids Res. 25:3389-3402,1997). As a result three open reading frames (ORFs) arranged in the samedirection were predicted (FIG. 2). The nucleotide sequence of the1650-bp ORF encoding CPMO is preceded by a partial ORFI (1173-bp) codingfor the C-terminus of an NtrC-ype transcriptional activator (Miranda, etal., The complete nucleotide sequence of the gInALG operon of Escherichacoil K12 15:2757-2770, 1987) and by a complete ORF2 (750-bp) coding fora homolog of the short-chain dehydrogenases/reductases (Jornvall, H., etal., Biochemistry 34: 6003-6013, 1995). The two intergenic regions are244-bp and 32-bp, respectively. The CPMO-encoding gene is referred tocpnB (cyclopentanone and B designates the second step of the degradationpathway, see FIG. 1) hereafter. In FIG. 5, the CPMO-encoding gene startsat nucleotide position 1822 and ends 3471 that does not include the stopcodon. Accordingly, the boundary of cpnA is 3507-4256. The partial openreading frame preceding cpnB is from 1-1174.

FIG. 1 has been adapted from Griffin, M., et al. (Griffin, M., et al.,Biochem. J. 129:595-603, 1972). The designated genes are: cpnA encodingcyclopentanol dehydrogenase; cpnB encoding cyclopentanone1,2-monooxygenase (CPMO). An alternative name for 5-valerolactone is5-pentanolide. Subsequent reaction steps are the formation of5-hydroxyvalerate, 5-oxovalerate, glutarate and finally acetyl CoA.

The amino acid sequence of the CPMO enzyme consists of 550 residues(FIG. 3). This sequence shows 36.5% identity and an additional 13.6%amino acid similarity with the 543-residue CHMO of Acinetobacter sp.strain NCIMB 9871. An equally related protein (549 amino acids; 37.3%identity and 12.4% similarity) is the putative steroid monooxygenase(STMO) of Rhodococcus rhodochrous (Morii, S., et al., GenBank accessionnumber AB010439, 1998). The latter enzyme carries out the oxidation ofprogesterone to produce testosterone acetate. A CLUSTAL alignment ofthese three sequences gave 24.6% positional identity (FIG. 3).

In FIG. 3, asterisks indicate identical amino acids, dots indicatedsimilar amino acids and dashes indicate gaps introduced to maximize thealignment. The amino-terminal peptide sequence confirmed by Edmandegradation is underlined. The locations of the consensus FADfingerprint sequences as described by Eppink et al. (Eppink, et al.,Prot. Sci. 6:2454-2458, 1997) are as indicated. The conserved GD motiffound in flavoprotein hydroxylases as a second FAD fingerprint is alsoindicated. Not shown is the DG motif of flavoprotein hydroxylases whichhas the sequence of chhhssDGxcSxhR. Lower case letters identify certainresidues types: h, hydrophobic residues, s, small residues, c, chargedresidues, and x, any residues. Note that a DG doublet is present in CPMOand STMO sequence.

A notable sequence motif present in CPMO and related proteins is theFAD-binding fingerprint (GXGXXG) that is similar to those found inflavoprotein hydroxylases (Eppink, et al., Prot. Sci. 6:2454-2458,1997). Flavoprotein hydroxylases (eg. phenol hydroxylase, the structureis now known; Enroth, C., et al., Structure 6:605-617, 1998) aremonooxygenases that catalyze the insertion of one atom of molecularoxygen into the substrate using NAD(P)H as electron donor. Theseproteins possess a conserved “Asp-Gly (DG)” motif for both FAD andNAD(P)H binding in between two fingerprint motifs for the FAD binding(fingerprint 1: GXGXXG; fingerprint 2: Gly-Asp [GD] motif). Sequencemotifs in CPMO, STMO and CHMO differ from those in flavoproteinhydroxylases by having a repeated GXGXXG motif (amino acids 24 to 33 and193 to 202 in CPMO numbering). The possibility that the second FADfingerprint in CPMO and related proteins fulfils a dual role of FAD andNADPH binding awaits structural determination of a representative memberof this family of proteins. It is reasonable to assume that a differentmechanism in catalysis is reflected in the motifs seen in the twoclasses of proteins.

Expression of cpnB Gene in E. coil

Two primers of the following sequence were synthesized to amplify thecpnB gene and the resultant 1.7-kb DNA fragment was cloned in the pSD80plasmid to yield pCMP201. Plasmid pSD80 is a third generation derivativeof the commercially available pKK223-3 vector (Pharmacia) that containsa tac promoter upstream of the multiple cloning site (MCS), an uncterminator sequence downstream of the MCS, and laclq elsewhere on theplasmid (Smith, S. P., et al., Biochemistry 35:8805-8814, 1996). Theprimers were: 5′-AAAAGGCCTG AACTTCAATT ATTTAGGAGA C-3′ (SEQ ID NO:3) and5′-AAAACTGCAG GAGTTGCACA ACAGAGTCTT AG-3′ with built-in StuI and PstIrestriction sites (underlined), respectively, to facilitate cloning atthe compatible sites (SmaI and PstI) of the pSD80 vector. Vent DNApolymerase (New England BioLabs, Beverly, Mass.) was used and theamplification conditions were 94° C. for 1 min, 50° C. for 1 min, and72° C. for 1 min, for 30 cycles. The amplified DNA fragment was purifiedfrom an agarose gel and digested with StuI and PsfI. One of theresulting recombinant plasmids was designated pCMP201. By DNA sequencingit was established that no mutation had been introduced in the cpnB geneduring PCR amplification.

FIG. 4 shows the production of a 60-kDa protein in a Coomassieblue-stained SDS-polyacrylamide gel of the crude protein extractprepared from E. coli JM109 (pCMP201) cells that were induced by 0.1 mMisopropyl-beta-D-thiogalactopyranoside (IPTG). The cells were induced atan absorbance (A600 nm) of 0.4 to 0.5 and the induction period was up to4 hr. The observed molecular mass was in agreement with the predictedsize of the 62-kDa CPMO. In the absence of IPTG, this protein band wasnot produced. Also, the CPMO enzyme activity was observed only in thosecells grown in the presence of IPTG. CPMO activity was assayed at 25° C.by measuring a decrease in absorbance at 340 nm in 50 mM phosphatebuffer (pH 7.8) containing 1 μmol of cyclopentanone, 0.2 μimol of NADPH,and the crude enzyme extract prepared from E. coil JMIO9 (pCMP201).These cells were cultivated in 100 ml of LB medium containing 100 μg/mIof ampicillin at 25° C. The IPTG-induced cells were harvested bycentrifugation, washed in 50 mM phosphate buffer (pH 7.2), resuspendedin 1/20 volume of same buffer, and sonicated by four-20 sec bursts witha Braun-Sonifier™ 250 apparatus. After centrifugation for 30 min at18,000×g and at 4° C., the supernatant was used for determination ofenzyme activity. One unit (U) of activity is defined as the amount ofenzyme required to convert 1 μmol of substrate in 1 min. Proteinconcentration was determined by the method of Bradford (Bradford, M. M.,Anal. Biochem. 72: 248-254, 1976). As a result the specific activity ofthe CPMO enzyme was found to be 0.28 U/mg. The specific activity of CPMOin the native Pseudomonas was reported to be 0.11 U/mg (Griffin, M., etal., Biochem. J. 129:595-603, 1972).

In FIG. 4, lane 1 has been loaded with extracts of IPTG-induced E. coliand lane 2 has been loaded with extracts of E. coli in absence of IPTG.M means molecular weight markers as indicated in kilo daltons. The arrowindicates the production of the desired 60-kDa protein.

Inactivation of cpnB Gene

Pseudomonas sp. strain HI-201 was constructed by chromosomalinactivation of the cpnB gene using a lacZ-Km^(r) cassette from themobilizable pKOK6.1 vector (Kokotek, W., et al., Gene 84: 467-471,1989). In pKOK6.1 the IacZ gene is promoterless and in addition toKm^(r) it is ampicillin resistant (Ap^(r)). The IacZ-Km^(r) cassette wasexcised as a PstI-fragment and inserted into the NsiI site within thecpnB gene in pCMP200, yielding pCMP202. Electroporation of this plasmidinto 9872 cells was carried out in the Gene Pulser™ (BioRads) and theparameters of electroporation were 2.5 kV, 25 uF and 200 ohm. The cellswere initially washed with 1 mM HEPES buffer and resuspended in 1 mMHEPES containing 10% glycerol. Km^(r) colonies were selected on LBplates containing Km (250 μg/mI). To select for double crossovermutants, a second screening on LB plates containing Ap (300 μg/ml) wascarried out. The inactivation of cpnB (FIG. 2), was confirmed by PCR.The resulting mutant HI-201 was found not to be able to grow oncyclopentanol or cyclopentanone as a sole carbon and energy source. Thisresult indicated that cpnB is essential for the degradaton ofcyclopentanol and it appeared that there was only one copy of the cpnBgene in strain 9872.

As expected of a flavoprotein the amino acid sequence of CPMO containsmotifs of FAD fingerprints similar to those found in flavoproteinhydroxylases.

Nucleotide Sequence Accession Number

The DNA sequence of the 4,281-bp SphI fragment has been submitted toDDBJ and assigned accession number AB022102. The release of this dataawaits the inventors' authorization.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof the appended claims.

1. An isolated DNA encoding a cyclopentanone monooxygenase (CPMO), or anenzymatically active portion thereof, the isolated DNA beingcharacterized by the ability to hybridize specifically with thecomplement of the DNA represented in SEQ ID NO:8 under stringenthybridization conditions.
 2. An isolated DNA, wherein it codes for acyclopentanone monooxygenase (CPMO), and contains: (a) the nucleic acidsequence of SEQ ID NO:8; (b) a sequence corresponding to said nucleicacid sequence in the scope of the degeneration of the genetic code; or(c) a sequence hybridizing under stringent conditions with the sequencefrom (1) or (2), and still coding for cyclopentanone monooxygenase(CPMO).
 3. An isolated DNA encoding a cyclopentanone monooxygenase(CPMO), or an enzymatically active portion thereof, said isolated DNAhaving SEQ ID NO:8.
 4. An isolated DNA expression vector encoding anenzymatically active cyclopentanone monooxygenase (CPMO) comprising aDNA characterized by a sequence as set forth in SEQ ID NO:8, or aportion thereof, said portion encoding said CPMO, in expressible form.5. A recombinant vector comprising the isolated DNA of any one of claims1 to 3, wherein the isolated DNA encodes cyclopentanone monooxygenase.6. The recombinant vector of claim 5, wherein the isolated DNA has anucleic acid sequence of SEQ ID NO:8 or which, due to the degeneracy ofthe genetic code, is a functional equivalent thereof.
 7. A recombinantvector, wherein it contains one or more copies of a recombinant DNAaccording to claim 1, 2 or
 3. 8. A recombinant vector according to claim4, 5, 6 or 7, wherein it is a prokaryotic vector.
 9. A recombinantvector according to claim 4, 5, 6 or 7, wherein it is a plasmid. 10.Biologically functional plasmid or viral DNA vector, which contains aDNA as defined in claim 1, 2 or
 3. 11. A host cell comprising arecombinant vector of any one of claims 4 to
 9. 12. A cell transformedwith a heterologous DNA expression construct encoding an enzymaticallyactive cyclopentanone monooxygenase (CPMO) comprising a DNAcharacterized by a sequence as set forth in SEQ ID NO:8, or a portionthereof, said portion encoding said CPMO, in expressible form.
 13. Acell as defined in claim 12, which is a prokaryotic cell.
 14. A cell asdefined in claim 12, which is E. coli.
 15. A purified cyclopentanonemonooxygenase (CPMO) having: (a) an amino acid sequence as set forth inSEQ ID NO:5; (b) an amino acid sequence encoded by a nucleic acidsequence as set forth in SEQ ID NO:8; or (c) an amino acid sequenceencoded by a nucleic acid sequence hybridizing to a nucleic acidsequence complementary to the. nucleic acid sequence of step b) aboveunder stringent conditions, said amino acid sequence encoded in step c)having a same activity as the amino acid sequence in a).
 16. Arecombinant cyclopentanone monooxygenase (CPMO) having an enzymaticactivity superior to the one naturally occurring.
 17. The recombinantcyclopentanone monooxygenase (CPMO) of claim 16, wherein said CPMO isprepared from Comamonas sp. NCIMB
 9872. 18. The recombinantcyclopentanone monooxygenase (CPMO) of claim 16, wherein said CPMO-has asequence as set forth in SEQ ID NO:5.
 19. The recombinant cyclopentanonemonooxygenase (CPMO) of claim 16, wherein said CPMO has an enzymaticactivity twice superior to that of a CPMO from a native Pseudomonas. 20.A method for growing cells in vitro in presence of cyclopentanol orcyclopentanone as sole source of carbon, said method comprising thesteps of: (a) transforming a cell with the expression construct of claim4, 5, 6, 7, 8 or 9; and (b) growing the cell of step a) under suitableconditions in a medium containing cyclopentanol or cyclopentanone as asole source of carbon.